Agglutination is primarily a chemical phenomenon in which surface interaction between macromolecules leads to crosslinking and the formation of a large complex, resulting in an increase in light scattering. The formation of this large macromolecular complex can be observed using turbidimetric, nephelometric or colorimetric detection.
Turbidimetric, nephelometric and colorimetric assays have the benefits of being quasi-homogeneous assays that do not require any separation or wash step. Due to their easy one-step procedure and their short turn-around times homogeneous immunoassays (HIA) are ideal candidates for the application in automated analyzers. Homogeneous immunoassays are routinely used in the clinical diagnostics for the quantitation of serum proteins, therapeutic drugs and drugs of abuse on clinical chemistry analyzers. So far, the standard method is the use of one main wavelength generating one calibration curve for the determination of analytes in turbidimetric, nephelometric and colorimetric assays.
Although an excellent assay performance can be achieved with HIA on the clinical chemistry analyzers, there are still patient samples with concentrations which are outside of the measuring range. As a consequence the samples have to be re-measured: after dilution in cases where the sample concentration exceeds the upper detection limit, or using higher sample volumes for samples with concentrations below the limit of detection. Re-measurements cause additional expenses and loss of time, both factors being critical for laboratories performing those assays. Therefore, an increase of the dynamic range would reduce the number of reruns.
State-of-the-art methods for addressing these issues are far from optimal—the known methods do not solve the problem of how to proceed with patient samples that are outside of the measuring range especially for automated systems. As a consequence the samples still have to be re-measured: after dilution in cases where the sample concentration exceeds the upper detection limit, or using higher sample volumes for samples with concentrations below the limit of detection. Re-measurements, so-called re-runs, cause additional expenses and loss of time, both factors being critical for laboratories performing those assays.
Furthermore, for several analytes two test systems are available for the measurement of high analyte concentrations and high sensitive test systems for the determination of a low analyte concentration. For example, for the analyte CRP (C-reactive protein) an additional high sensitivity C-reactive (hs-CRP) immunoassay for cardiovascular risk assessment exists (Tina-quant® Cardiac C-reactive protein (Latex) high sensitive (Roche Diagnostics GmbH)) for the determination of low analyte concentration.
As a result, there is a high need for increasing of the sensitivity and extending of the dynamic range in photometric assays.